We have recently developed model systems to study the mammalian cellular function of Rho GTPases (e.g., Cdc42) and the Wiskott-Aldrich syndrome proteins (e.g., WASP, N-WASP, WAVEs) - two interacting families of proteins that integrate incoming cell surface signals to mediate cytoskeletal change. WASPs are cytoplasmic proteins that when activated by Rho-family GTPases (Cdc42/Rac) and phosphoinositides directly bind to the Arp2/3 complex, resulting in actin assembly. In this context, coordination of cell shape through cytoskeletal change is required for such diverse properties as cell-cell contact, lymphocyte activation, and chemotaxis. WASP activation is also regulated by interaction with several other proteins including the WASP-interacting protein (WIP), Sh3 domain proteins (e.g. Nek) and Srk family kinases (e.g., Hck). We have employed gene-targeting to generate mice deficient for the Rho-family GTPase Cdc42 as well as several WASP family members: WASP, N-WASP, and WAVE-2. N-WASP- and WAVE-2- deficiencies result in early embryonic lethality whereas WASP-deficient (WKO) mice are viable and fertile, with lymphocytes that develop normally but have signaling and cytoskeletal abnormalities. We have also generated ES cells clones that contained an activated WASP knock-in mutation associated with the recently described novel immunodeficiency, X-linked Neutropenia (XLN). Because N-WASP and WAVE-2 deficiencies were not viable, selective targetings of these alleles were required to assess the role of these proteins in lymphocytes. To this end, we have generated mice with N-WASP that can be conditionally inactivated in leukocytes, and we are currently generating mice with similar mutations in WAVE2. We have generated WASP/N-WASP double knock-out (DKO) mice in T cells and, in collaboration with Dr. Raif Geha (Project 1), WASP/WIP DKO mice;both strains are associated with more severe signaling and cytoskeletal abnormalities in T cells when compared to WKO mice. This collection of novel reagents, in which the various WASP-family members can be inactivated, singularly or in combination and in either cells or mice, provides a powerful basis for our ongoing goals of dissecting the roles of WASP family members in leukocyte function. We propose: A1) To dissect the unique role of WASP and the combined role of WASP/N-WASP in leukocyte development and function. A2) To define the role of constitutively-active mutations in WASP function in T cells.